Antioxidant activity of methanol extracts of Enhalus acoroides and Thalassia hemprichii from the coastal water of Carmen, Agusan Del Norte, Philippines

178 Tangon et al. Int. J. Biosci. 2020 RESEARCH PAPER OPEN ACCESS Antioxidant activity of methanol extracts of Enhalus acoroides and Thalassia hemprichii from the coastal water of Carmen, Agusan Del Norte, Philippines Efren Tangon1*, Oliva P. Canencia1, Romeo M. Del Rosario1, Elvinia R. Alivio2 1Department of Science Education, University of Science and Technology of Southern Philippines, Cagayan de Oro City, Philippines 2College of Education, Mindanao State University, TawiTawi, Philippines Key words: Seagrass, Antioxidants, Tropical, Phenolics, Flavonoids. http://dx.doi.org/10.12692/ijb/16.6.178-184 Article published on June 29, 2020 Abstract Antioxidants play an important role in apoptosis, gene expression, and ion transportation and may reduce the risk of many diseases by protecting the cells against the effect of free radicals. The antioxidant potential of methanolic extracts of the seagrasses Thalassia hemprichii and Enhalus acoroides collected from the coastal water of Carmen, Agusan Del Norte, Philippines was determined using Aluminum chloride complex forming assay for total flavonoid content, Folin–Ciocalteu reagents with analytical grade gallic acid as the standard for the total phenolic content, DPPH, ABTS and FRAP. The results showed that methanol extract of Thalassia hemprichii had the highest content of total phenolics and flavonoids which values were 2.651 and 2.734 mgGA/g respectively. The strongest free radical scavenging activity (DPPH) of the extracts was recorded by seagrass Enhalus acoroides which value was 0.301 mgtrolox/g. While the methanol extracts of Thalassia hemprichii recorded the maximum radical cation decolorization power (ABTS) and Ferric ion reducing antioxidant power (FRAP) which values were 0.252 and 1.119 mgtrolox/g respectively. The antioxidant activity determined by DPPH, ABTS and FRAP demonstrated a strong linear relationship with the phenolics and flavonoids. The results suggested that the sea grasses Thalassia hemprichii and Enhalus acoroides have strong antioxidant potential and could be a source of natural antioxidant compounds. * Corresponding Author: Efren Tangon  efrenyutango[email protected] International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 16, No. 6, p. 178-184, 2020 179 Tangon et al. Int. J. Biosci. 2020 Introduction Seagrasses are submerged flowering plants found in shallow marine waters, such as bays and lagoons and along the Gulf of Philippines. Nineteen seagrass species were found from more than 529 sites in the Philippines. In relation to seagrass as a resource in need of protection, its status as such is yet largely unknown, becoming a focus of scientific inquiry only in the last 30 years and as an object of conservation, only in the last 15 years (Fortes, 2012). The vast biodiversity and sensitivity to changes in water quality inherent in seagrass communities makes seagrasses an important species to help determine the overall health of coastal ecosystems. Seagrasses grown in the tropical climate like Philippines are expected to bask in strong ultraviolet radiation. This circumstance can causeincrease levels of reactive radical species. To reduce or protect, they may change their metabolism and stimulate them to produce some active compounds, therefore tropical seagrasses are estimated to possess a large number of active compounds. The secondary compounds like polyphenols and flavonoids are the key factors that are involved in the adaptation to changing biotic and abiotic environments and also mainly for the defense mechanism. Natural antioxidants and their association with health benefits have gained unprecedented attention in recent years. They have multiple functions in biological systems and mainly defense against oxidation that produce free radicals in food, chemicals and in living systems. During normal cellular activities, various processes produce reactive oxygen species (ROS) inside the cell, which can damage the cellular components such as lipids, proteins, and DNA, when produced at high rates. The major action of antioxidants in cells is to prevent the damage caused by the action of reactive oxygen species (Kanna et al., 2010). The aim of this study was to assess the antioxidant potential of the seagrasses Enhalus acoroides and Thalassia hemprichii of Butuan Bay, Agusan Del Norte, Philippines. Materials and methods Chemicals and reagents Chemicals and reagents used in this experiment were methanol, NaNO2, AlCl3, NaOH, FC reagent, Na2CO, FRAP reagent, ABTS reagent and DPPH reagent. All the chemicals were analytical grade and all chemicals were obtained from Elmar Marketing, Iligan city and Merteflor, Cagayan De Oro city, Philippines. Sample collection The seagrasses that were used in this study were collectedfrom the coastal water of Carmen, Agusan Del Norte, Philippines. Geographically, Carmen is located at 9°00′N 125°16′E. The collected seagrasses was washed thoroughly with tap water to remove all sand particles and epiphytes then brought to the chemistry laboratory of University of Science and Technology of Southern Philippines (USTP) at Lapasan, Cagayan De Oro city, Misamis Oriental and shade dried at room temperature. The dried seagrass samples were then grounded on the mixer and stored in the refrigerator individually in airtight containers for further use. Methanolic extraction About 25.00 g of the sample was soaked in 95% methanol. A minimum volume of 200 ml was used to soak the sample. The soaking took about 48 hours. After 48 hours, the sample was filtered using Whatman filter paper. Then another 100 ml of methanol was used for the second soaking. Then after an hour, it had been filtered again. And for the soaking, another 100 ml of methanol was used, after which, the filtrate was then placed in the refrigerator for proper storage. Total phenolic content The total phenolic content of all the formulations of seagrasses was determined by using FolinCiocalteu method. 0.5 ml of the plant extract was placed in a 25 ml vial and 4.5 ml of distilled water was added. 0.5 ml of FC reagent was mixed with the solution and 10 ml 0f 7% Na2CO3 was added. The FC reagent was prepared by dissolving about 0.0166 g of Gallic Acid monohydrate with absolute methanol and diluted to 180 Tangon et al. Int. J. Biosci. 2020 50 ml. 2.5 ml distilled water was added then to make a 12.5 ml solution. The solution was incubated for 90 minutes and then the absorbance was read at 750 nm using UV-VIS Spectrophotometer. The total phenolic content of the sea grass was calculated as gallic acid equivalents (mgGAE/g). All the experiments were performedin triplicate. Total flavonoid content Aluminum chloride complex forming assay was used to determine the total flavonoid content of the extracts. Quercetin was used as standard and flavonoid content was determined as quercetin equivalent. 1 ml of the 1,000 ppm of plant extract was placed in a clean vial. 5ml of absolute methanol was added and followed by 300 µL or 0.3 mL of 5% NaNO2. The mixture was allowed to stand for 5 minutes at room temperature. 600 µL or 0.600 mL 10% AlCl3 was added and allowed it to stand again for 6 minutes at room temperature. 2 ml of 1 mM NaOH and 1.10 ml of absolute methanol were added and the mixture was incubated for 20 minutes at room temperature and then the absorbance was read at 510 nm using UV-VIS Spectrophotometer. Total flavonoid content was calculated as quercetin equivalents (mgQE/g). All the procedures were performed in triplicate. Scavenging activity (DPPH) assay The free radical scavenging activities of the extracts was determined by using 2, 2Diphenyl-1picrylhydrazyl (DPPH) free radical scavenging method. 0.2 ml of the 1,000 ppm of seagrass extract was placed in a clean vial and 5.8 ml of 0.01 mM DPPH reagent was added. The DPPH reagent was prepared by dissolving about 0.0250 g of Trolox with absolute ethanol and diluted to 100 ml in a volumetric flask. The mixture was then incubated for 30 minutes in the dark at room temperature and the absorbance was read at 517 nm using UV-VIS spectrophotometer. ABTS radical cation decolorization power The ABTS radical cation decolorization power was determined according to the method described by Irondi et al., (2012) with slight modification. 0.2 ml of the 1,000 ppm of plant extract was placed in a clean vial and 5.8 ml of the ABTS reagent was added. The ABTS reagent was prepared by dissolving about 0.0250 g of Trolox with absolute ethanol and diluted to 100ml in volumetric flask. The mixture was then incubated for 6 minutes at room temperature and the absorbance was then read at 734 nm using UV-VIS spectrophotometer. Ferric reducing antioxidant power (FRAP) The property of the methanolic extract was determined by assessing the ability of the extracts to reduce FE as described by Irondi et al., (2012) with slight modification. 4.0 ml of the 1,000 ppm of plant extract was placed in a clean vial and 6.0 ml of the FRAP reagent was added. The FRAP reagent was prepared by dissolving about 0.0139 g of FeSO4.7H2O with distilled water and diluted to 100 ml. The mixture was then incubated in a water bath at 370 C and the absorbance was read then at 593 nm using UV-VIS spectrophotometer. Statistical analysis Three replicates of each sample were used for statistical analysis and the values were reported as mean + SD. Pearson’s correlation analysis was carried out using Minitab, version 17 software to study the relationship between antioxidant activities and total phenolic, flavonoid content. Results and discussion Table 1 showed the results of total phenolics and flavonoids content of the seagrasses. The maximum total phenolic content was recorded by the seagrass Thalassia hemprichii 2.651+ 0.001 followed by Enhalus acoroides 0.201 + 0.028 mgGA/g. Seagrasses are a rich source of phenolic substances, including phenolic acids, sulphated phenolic acids, flavones, condensed tannins and lignins, but not hydrolyzable tannins. The phenolic acids that predominate in the seagrasses also occur widely in land plants, but gallic acid was detected in a greater percentage of seagrasses (Zapata et al., 2019). 181 Tangon et al. Int. J. Biosci. 2020 Phenolic compounds in plants play an important role in pigmentation, growth, reproduction, resistance against pathogens, defense mechanism as well as protecting plants from deleterious effects of ultraviolet radiation and oxidants. The total phenolic content of a plant is an important parameter for their antioxidant properties. Table 1. Total phenolic, total flavonoid and antioxidant activities of the seagrasses. Sea Grass TPC (mg GA/g) TFC (mg Q/g DPPG (mg Trolox/g) ABTS (mg Trolox/g) FRAP (mg Trolox/g) E. acoroides 0.201+ 0.028 1.805+ 0.274 0.301 +0.003 0.007 + 0.000 0.063 + 0.000 T. hemprichii 2.651+ 0.001 2.734+ 0.047 0.189 +0.001 0.162 + 0.001 1.119 + 0.019 Values are means + SD for 3 determinations. The highest total flavonoid content was recorded on the seagrass Thalassia hemprichii 2.734+ 0.047 and Enhalus acoroides recorded 1.805 + 0.274 mgQ/g. Forty three species of seagrasses were exclusively studied and identified that all contained either flavones and/or phenolic acid sulfates. Among the 12 genera examined, five (Zostera, Phyllospadix, Enhalus, Thalassia and Halophila) had sulfated flavones (Subhashini et al., 2013). It has been reported that flavonoids are free radical scavengers that prevent oxidative cell damage, and have strong anticancer activities (Pourmorad et al., 2006; Ugwu et al., 2013) and they might induce mechanism that affects cancer cells and inhibit tumor invasion (Rafat et al., 2008). These activities could be attributed to their ability to neutralize and quench radicals (Pourmorad et al., 2006; Omale and Okafor, 2008; Ugwu et al., 2013). It can also be due to their redox properties, presence of conjugated ring structures and carboxylic group which have been reported to inhibit lipid peroxidation (Rice-Evans et al., 1995). In plant systems, flavonoids help in combating oxidative stress and act as growth regulators. Table 2. Correlation between total phenolic content, total flavonoid content and antioxidant assays. Antioxidant assay DPPH assay ABTS assay FRAP assay R2 P-value R2 P-value R2 P-value TPC 0.99 0.000 0.99 0.000 0.99 0.00 TFC 0.66 0.048 0.62 0.064 0.66 0.051 The effect of antioxidants on DPPH radical scavenging is thought to be due to hydrogen donating ability. DPPH is a stable free radical and it accepts an electron or hydrogen radical to become a stable diamagnetic molecule. When a DPPH solution is mixed with a substrate acting as a hydrogen atom donor, a stable non-radical form of DPPH solution is mixed with a substrate acting as a hydrogen donor, a stable non radical from DPPH is obtained with simultaneous change of the violet color to pal Hence, DPPH (1,1-diphenyl-2-picrylhydrazyl) has been used extensively as a free radical to evaluate reducing substances and is a useful reagent for investigating the free radical scavenging activities of compounds. The maximum free radical scavenging activities (DPPH) of the extracts as shown on Table 1 was recorded by Enhalus acoroides 0.301 +0.003 followed by Thalassia hemprichii 0.189 +0.001 mgtrolox/g. Table 2 showed the results of correlation between total phenolic content, total flavonoid content and antioxidant assays. There was a strong relationship between total phenol content, total flavonoid content and antioxidant activity determined by DPPH radical scavenging which values R2=0.99, P value = 0.000 and R2=0.66, P value=0.048 respectively. Phenol in the form of condensed tannin (proanthocyanidins) was found as the main phenolic compound E. acoroides (Kannan et al., 2010). 182 Tangon et al. Int. J. Biosci. 2020 ABTS or 2,2-azino-bis (3-ethylbenzothiazoline-6sulphonic acid) is a chemical compound used to observe the reaction kinetics of specific enzymes. ABTS is converted to its radical cation by addition of sodium persulfate. This radical cation is blue in color and absorbs light at 734 nm. The ABTS radical cation is reactive towards most antioxidants including phenolics, thiols and Vitamin C. During this reaction, the blue ABTS radical cation is converted back to its colorless neutral form. The reaction may be monitored spectrophotometrically. This assay is often referred to as the Trolox equivalent antioxidant capacity (TEAC) assay. The reactivity of the various antioxidants tested is compared to that of Trolox, which is a water-soluble analog of vitamin E. The strongest ABTS radical cation decolorization power was recorded by the sea grass Thalassia hemprichii with values was 0.162 + 0.001mgtrolox/g followed by Enhalus acoroides 0.007 + 0.000 mg Trolox/g. There were a strong relationship between total phenol content, total flavonoid content and antioxidant activity determined by ABTS radical cation decolorization power which values R2=0.99, P value = 0.000 and R2=0.62, P value=0.064 respectively. FRAP, also Ferric ion reducing antioxidant power is an antioxidant capacity assay that uses Trolox as a standard. The FRAP assay was first performed by Benzie and Strain (1996). The method is based on the formation of O-Phenanthroline-Fe2+ complex and its disruption in the presence of chelating agents. This assay is often used to measure the antioxidant capacity of foods, beverages and nutritional supplements containing polyphenols. It measures the ability of antioxidants in plasma or in foods to reduce the ferric component (Fe3+) of a ferric tripyridyltriazine (Fe3+-TPTZ) complex (which is contained in the FRAP reagent) to the ferrous form (Fe2+). During this reaction which takes place at a low pH, the reduction of ferric iron (Fe3+) to the ferrous form (Fe2+) is accompanied by the formation of a blue color which can be measured at an absorption maximum of 593nm using a spectrophotometer. The strongest Ferric ion reducing antioxidant power was recorded by the seagrass Thalassia hemprichii with value of 1.119 mg Trolox/g and followed by Enhalus acoroides 0.063 mg Trolox/g. There were a strong relationship between total phenol content, total flavonoid content and antioxidant activity determined by (FRAP) Ferric ion reducing antioxidant power which values R2= 0.99, p value = 0.000 and R2= 0.66, P value=0.051 respectively. The results of this study are in line with the previous researches conducted by Kannan et al., (2010a), Kannan et al., (2010b), Gavin and Durako, (2011), Sanatoso et al.,(2012), Kannan et al., (2013), Athiperumalsami et al., (2008) and Baby et al., (2017) all showed that the antioxidant activity determined by DPPH, ABTS and FRAP demonstrated a significant positive linear correlations with their phenolics and flavonoids. Conclusion This study showed that the seagrasses were rich sources of natural antioxidants. The total content of polyphenols and flavonoids in the methanol extracts of the seagrass species positively correlated with their antioxidant properties. The results obtained suggest that the strong antioxidant properties of the seagrass species could play an important role in the food and pharmaceutical industries and realizing the true potential of seagrass meadows therefore requires an international cooperation to conserve and protect seagrass. References Athi Perumalsami T, Rajeswari VD, Poorna SH. 2010. Antioxidant activity of seagrasses and seaweeds. Botany 53(3), 251–7. Baehaki A, Supriadi A, Pratama MC. 2016 Antioxidant Activity of Methanol Extract of Halodule uninervis Seagrass from the Coastal of Lampung, Indonesia. Research Journal of Pharmaceutical, Biological, and Chemical Sciences, 173-177 p. Benzie IF, Strain JJ. 1996. The Ferric Reducing Ability of Plasma (FRAP) as a measure of “Antioxidant Power”: The FRAP assay. Journal 183 Tangon et al. Int. J. Biosci. 2020 Analytical Biochemistry 239(1), 70-6. Chaillou LL, Nazareno MA. 2006. New method to antioxidant activity of polyphenols. Journal of Agriculture and Food Chemistry 54(22), 8397-402. Camposs AM, Lissi EA. 1996. Kinetics of the reaction between 2,2′-azinobis (3ethylbenzothiazoline-6-sulfonic acid (ABTS) derived radical cations and phenols. International Journal of Chemical Kinetics 29(3), 219-24. Cannac M, Ferrat L, Barboni T. 2007. The influence of tissue handling on the flavonoid content of the aquatic plant Posidonia oceanica. Journal Chemical Ecology 33(5), 1083-8. Dumay O, Costa J, Desjobert JM, Pergent G. 2004. Variations in the concentrations of phenolic compounds in the seagrass Posidonia oceanica under conditions of competition. Phytochemistry 65, 32113220. Fortes MD. 2013. A Review: Biodiversity Distribution and Conservation of Philippine Seagrasses. Philippine Journal of Science 142, 95-111, Special Issue. Gringnon-Dubois M, Rezzonico B, Alcoverro T. 2012. Regional scale patterns in seagrass defenses: Phenolic acid content in Zosteranoltii. Estuarine, Coastal and Shelf Science 114, 18-22. Gavin NM, Durako MJ. 2011. Localization and antioxidant capacity of flavonoids from intertidal and subtidal Halophila johnsonii and Halophila decipiens. Aquatic Botany 95, 242-247. Green EP, Short FT. 2003. World Atlas of Seagrasses. Berkeley, USA: University of California Press. Kahkonen MP, Hopia AI, Vuorela HJ, Rauha JP, Pihlaja K, Kujala TS. 1999. Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural and Food Chemistry47, 3954-3962. Kannan RR, Arumugam R, Anantharaman P. 2010.In vitro antioxidant activities of ethanol extract from Enhalus acoroides (L.F.) Royle. Asian Pacific Journal of Tropical Medicines 3(11), 898-901. Kannan RR, Arumugam R, Thangaradjou T, Anantharaman P. 2013. Phytochemical constituents, antioxidant properties and p-coumaric acid analysis in some seagrasses. Food Research International 54(1), 1229-36. Kannan RR, Arumugam R, Meenakshi S, Anantharaman P. 1984. Thin layer chromatography analysis of antioxidant constituents from seagrasses of Gulf of Mannar Biosphere Reserve, South India. International Journal Chemistry and Technology. Seagrasses. Aquatic Botany 20, 351-7. Kuo J, Den Hartog C. 2001. Seagrass taxonomy and identification key. In: SHORT, F.T AND COLES, R.G., eds. Global Seagrass Research Methods. Amsterdam, Elsevier 31-58. Larson RA. 1999. Plant defenses against oxidative stress. Archives Insect Biochemistry and Physiology 29(2), 175-86. Libin B, Sankar TV, Chandramohanakumar N. 2017. Changes in phenolic compounds in sea grass against change in the ecosystem. Phytojournal.Com/archives/2017/6(3), PartL./6-362-162.pdf. Molyneux P. 2004. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin Journal of Science and Technology 26(2), 211-9. Pulido R, Bravo L, Saura-Calixto F. 2000. Antioxidant Activity of Dietary Polyphenols as determined by a modified ferric reducing/antioxidant power assay. Journal of Agriculture and Food 184 Tangon et al. Int. J. Biosci. 2020 Chemistry 48(8), 3396-402. Ragupathi Raja Kannan R, Rajasegaran A, Thirunavukarasu T. 2013. Phytochemical constituents, antioxidant properties and p-coumaric acid analysis in some seagrasses. Food Research International. Rice-Evants C, Miller NJ, Bolwell GP, Bramley PM, Pridham JB. 1995. The relative antioxidants activities of plant-derived polyphenolic flavonoids. Free Radical Research 22, 375-383. McMillian C. 1984. The condensed tannins (proanthocyanidins) in seagrasses. Aquatic Botany 20, 351-357. Molyneux P. 2004. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin Journal of Science and Technology 26(2), 211-9. Neelima CSS, Seenivasan R. 2015. DPPH radical scavenging activity of selected Seagrasses from South East Coast of India. International Journal of Advanced Research 3, 950 – 956. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, RiceEvans C. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicines 26(9), 1231-7. Santoso J, Anwariyah S, Rumiantin RO, Putri AP, Ukhty N, Yoshie-Stark Y. 2012. Phenol Content, Antioxidant Activity and Fibers profile of four tropical seagrasses from Indonesia. Journal of Coast Development Research 15(2), 189-96. Somanah MJ, Abdoulrahman N, Bhagooli R. 2012. Assessment of phenol content and antioxidant activities of shallow-water macroalgae from Mauritius. University of Mauritius Research Journal, 18A, 28-53. Sullivan ML. 1994. The taxonomy of "seagrasses" surveyed from the higher taxa down through to the family level [Online]. Florida International University. Available from: http://www2.fiu.edu/~seagrass/class/bot5647/maur een.htm. Szabo MR, Iditoiu C, Chambre D, Lupea AX. 2007. Improved DPPH Determination for Antioxidant Activity Spectrophotometric Assay. Chemistry Papers 61(3), 214-6. Villano D, Fernandez-Pachon MS, Moya ML, Troncoso AM, GarciaParilla MC. 2007. Radical scavenging ability of phenolic compounds towards DPPH free radical. Talanta 71, 230–235. Zapata O, McMillan C. 1979.Phenolic acids in seagrasses. Aquatic Botany 307-17.